The ubiquitin-proteasome system is well known as a proteolytic pathway possessed by cells. In this reaction system, linear chains comprising several molecules of a small protein called ubiquitin are attached to denatured proteins or abnormally folded proteins. The ubiquitin chains can mark the proteins for degradation, which are in turn recognized and destroyed by proteasome, a proteolytic machine. This system performs the removal of intracellular abnormal proteins. The ubiquitin-proteasome system, however, is not always a system intended only for the quality control of intracellular proteins. The system controls various cell functions by degrading even structurally or functionally normal proteins according to cell states of the moment and thereby suppressing their activities. The ubiquitin-proteasome system has been found so far to control the abundances of many proteins. These proteins have diverse functions, such as control of cell cycle, regulation of gene expression, stress response, and DNA repair. In this way, the ubiquitin-proteasome system controls many life phenomena exhibited by cells and is thus considered essential for the maintenance of normal cell activity. Therefore, the hypofunction of this proteolytic system has a critical impact on cells. The abnormal intracellular accumulation or aggregation of proteins is observed in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. It has been suggested that the onset of these diseases is caused by the abnormal function of the ubiquitin-proteasome system. Since the ubiquitin-proteasome system is also involved in cell cycle, DNA repair, and the like, the disruption of this system is also known to induce the malignant transformation of cells. In this way, the ubiquitin-proteasome system is responsible for the control of many important life phenomena. Thus, the development of an approach of precisely and conveniently measuring this reaction system would make a significant contribution not only to the elucidation of mechanisms underlying life phenomena exhibited by cells but also to the development of therapy or drugs for diseases induced by abnormality therein.
Protein degradation by the intracellular ubiquitin-proteasome system has previously been measured using a biochemical approach such as Western blotting. In this approach, many cells are collectively destroyed, and their components are recovered. Protein analytes contained therein are electrophoretically separated according to molecular weights and further detected by staining using specific antibodies. As a result, the existing levels of the proteins or proteolytic activities on the proteins can be measured. Unfortunately, this approach requires complicated operation and much time and does not permit assay in individual living cells.
In recent years, great development has been brought about in techniques of applying luciferin and luciferase involved in the bioluminescent reaction seen in firefly or the like, or fluorescent proteins obtained from Aequorea victoria or the like, to probe reagents for monitoring intracellular molecular dynamics. Such techniques have been coupled with the advance of microscopic imaging techniques to thereby popularize approaches of spatiotemporally visualizing and measuring a particular physiological activity in cells. This approach has also allowed the proteolytic activity of the proteasome to be measured in living cells. In this approach, a protein analyte is fused with luciferase or a fluorescent protein and used as a probe reagent (Patent Literature 1). When this protein exists in cells, the luminescence or fluorescence which is a label is observed; however, when this protein is degraded by proteasome, the fused luciferase or fluorescent protein is degraded together with the protein, resulting in no observable luminescence or fluorescence. Thus, change in the intensity of this light can be monitored in order to measure a proteolytic activity on this protein. The degradation of proteins such as IκBα, p27, p53, or HIF-1α has been measured so far by this approach (Patent Literature 2 and Non Patent Literatures 1 to 3). Since one type of label, such as luciferase or a fluorescent protein, is used in these probe reagents, the analyte is measured at only one wavelength of luminescence or fluorescence. Therefore, the intensity of the light is influenced by factors independent of proteolytic activity, such as the nonuniform distribution or expression level of the probe reagent, cell or tissue morphology, quenching caused by fading or the like, or nonuniform illumination with excitation light, thereby causing difficulties in quantitative measurements. In addition, when light quantity is decreased by increase of proteolytic activity, the insufficient sensitivity of a detector or a reduced S/N ratio is disadvantageously caused, thereby making precise measurements difficult.
The undesired influence of protein degradation-independent factors in the photometric method using only one wavelength can be canceled by measuring luminescence or fluorescence at two wavelengths and determining the ratio of intensities. In the approach of Davis et al., a protein analyte IκBα fused to click beetle-derived luciferase (CBG68) exhibiting green luminescence was expressed in cells. At the same time, click beetle-derived luciferase (CBR) exhibiting red luminescence was also expressed in these cells. CBG68 exhibits increase or decrease in the amount of its luminescence depending on proteasomal degradation activities on IκBα. On the other hand, the luminescence of CBR is insusceptible to proteasomal degradation and, as such, was used as a control. Davis et al. measured luminescence in a cell group cultured in a multi-well plate, and measured an IκBα-proteolytic activity as a green/red ratio of luminescence intensities in order to correct the difference in the amount of luminescence derived from, for example, different numbers of cells among wells (Non Patent Literature 4). This approach improves quantitative performance compared with the 1-wavelength photometric method, but disadvantageously, can hardly equalize the expression level ratio between two probe molecules, i.e., luminescence intensity ratio, among cells because these two probe molecules are individually expressed. In addition, the luciferase or fluorescent protein used as a label might be nonuniformly distributed in cells, depending on its properties, and thus differ in its localization among the cells. These are responsible for the degraded accuracy of proteolytic activity assay particularly at a single-cell level.